Peroxidase Conjugation by Periodate Method

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Tony Rook
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Peroxidase Conjugation by Periodate Method

Please find the link for the following protocol:

Peroxidase Conjugation by Periodate Method
Originally posted by Nanci Donacki at:

http://www.protocol-online.org/prot/Detailed/440.html

Reagents

1. Horseradish peroxidase
2. Sodium periodate, 0.1M, freshly made
3. Ethylene glycol
4. Sephadex G-25
5. Sodium acetate buffer, 0.001M, pH 4.2
6. IgG
7. Carbonate:Bicarbonate Buffer, 1.0 M, pH 9.5
8. Sodium tetraborate (4 mg/ml) freshly made.
9. PBS

Procedure

1. Dissolve 16 mg HRP in 4 ml diH2O and 0.8 ml of sodium
periodate.
2. Stir for 20 minutes, RT
3. Add 6 drops ethylene glycol
4. Stir for 5 minutes, RT
5. Filter over a G-25 Sephadex column equilibrated with 1mM
acetate
6. Pool colored fractions and divide into two equal parts.
7. To the first part, add 14 mg IgG
8. To the second part, add 10 mg IgG.
9. To both add 1M carbonate buffer, dropwise, to raise the pH
to 9.0-9.5
10. Stir for 2 hours at room temperature
11. To both add 0.2 ml sodium tetraborate
12. Incubate for 2 hours at 2-8oC
13. Dialyze against PBS.
14. Separate on ACA-44 column, eluted with PBS
15. Pool fractions and add 10 mg/ml PBS